The mosquito-borne flavivirus, dengue, is estimated to cause in each year 100 million cases of dengue fever (DF), 500,000 cases of dengue hemorrhagic fever (DHF) and 25,000 deaths, with 2.5 billion people at risk (Monath, T. P. 1994 PNAS USA 91:2395-2400). Although a successful vaccine against the prototypical flavivirus, yellow fever (YF) virus, has been in use since the 1930s and vaccines to two other flaviviruses, Japanese encephalitis (JE) virus and tick-borne encephalitis (TBE) virus are currently available, there is as yet no dengue vaccine approved for use (Cardosa, M. J. 1998 Brit Med Bull 54:395-405).
Dengue virus has a typical flavivirus genome structure, as described in FIG. 2A. The structural proteins, C, prM (M) and E, are involved in packaging, export and subsequent entry. The non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 include an RNA-directed RNA polymerase, and a protease function involved in cleaving certain positions of the long viral polyprotein which contains all the viral genes (Chambers, T. J. et al. 1990 Ann Rev Microbiol 44:649-88; Rice, C. M. 1996 In: Fields Virology, 3rd ed. Philadelphia, Pa. Lippincott-Raven Publishers, p. 931-996).
The four serotypes of dengue virus (“1” through “4”) share approximately 60%-74% amino acid residue identity with one another in the E gene (Thomas, C. J. et al. 1990 Ann Rev Microbiol 44:649-88) and induce cross-reacting antibodies (Heinz, F. X. 1986 Adv Vims Res 31:103-168). However, neutralizing antibodies to the structural proteins of one serotype of dengue typically not only fail to provide protection against other serotypes, but appear to cause the enhanced replication of virus seen in dengue hemorrhagic fever, which is generally seen upon reinfection by dengue virus of a different serotype. This antibody-dependent enhancement of infection (ADE), which is believed to be mediated by enhancement of viral uptake by macrophages (Morens, D. M. 1994 Clin Infect Dis 19:500-512) complicates dengue vaccine development, since an inadequate or modified immunogen may contribute to disease, rather than prevent infection (Halstead, S. B. 1988 Science 239:476-481).
Two strategies suggest themselves for circumventing the problems caused by cross reacting antibodies against the major structural proteins, prM and E. One strategy is to immunize with multiple strains of dengue virus to elicit high affinity, neutralizing antibodies against the multiple dengue serotypes. At least one vaccine to do this (using dengue vaccine candidates DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK 30/F3, and DEN-4 PDK48) has been in clinical trials (Bhamarapravati, N. and Sutee, Y. 2000 Vaccine 18 Suppl 2:4447; Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188). A second strategy is to induce immunity only to viral proteins other than prM and E. Several studies have shown that the nonstructural glycoprotein NS1 can play an important role in protection against dengue. Mice immunized with purified dengue-2 NS1 protein injected intramuscularly and boosted after 3 days and two weeks were protected from developing lethal dengue encephalitis upon subsequent challenge with dengue-2 virus (Schlesinger, J. J. et al. 1987 J Gen Virol 68:853-857). Similarly, mice immunized with recombinant vaccinia virus expressing authentic NS1 (Falgout, B. et al. 1990 J Virol 64:4356-4363) were protected against the development of dengue-4 virus encephalitis when challenged by intracerebral injection. Inoculation of mice with specific combinations of MAbs directed against dengue-2 NS1 (Henchal, E. A. et al. 1988 J Gen Virol 69:2101-2107) also protects against lethal virus encephalitis upon intracerebral dengue-2 challenge. Other nonstructural proteins are also immunogenic and may participate in eliciting protection (Brinton, M. A. et al. 1998 Clin Diagn Virol 10:129-39).